The polymerase chain reaction - Journal of Chemical Education (ACS

Apr 1, 1993 - Techniques in using polymerase chain reactions (PCR) especially for applications in lab activities geared toward understanding HIV, DNA ...
1 downloads 0 Views 8MB Size
Download PDF
concepts in biochemistry

WILLIAM M. SCOVELL edited by Bowling Green State University Bcwling Green, OH 43403

The Polymerase Chain Reaction William C. ~ i m m e r 'and Juanita M. Villalobos National Institutes of Health. National Institute of Allergy and Infectious Diseases, Laboratory of ~rnmunore~ui~tion, Bethesda, MD 20892

.~.

amounts of single copy genes from genomic DNA (2).PCR requires two synthetic oligonucleotides, or primers, of ~

The -~~~nolvmerase chain reaction (PCR) is a n enzvmatic process employed to amplib specific sequences of r j or ~ KNA.PCR consistsof three s t c ~ w i s o~erations, e which arc sequentially performed: ~

about 20 base pairs (bp) each; a DNApolymerase; the four nucleotide triphosphates (dNTPs: dATP, dCTP, dGTP, and dTTP); magnesium ion; and the template DNA that is to be amplified. The primers are complementary to sequences that flank the target sequence on opposite strands of the template DNA. They are oriented with their 3' ends directed toward each other so that synthesis by DNA polymerase extends across the DNA segment between them. The first reaction step involves denaturation of the template DNA a t a temperature of -95 'C. This is performed with a molar excess of the primers, dNTPs, and M e , in the presence of the template DNA. The temperature is then lowered to between 37-55 'C. The concentration dif-

1. DNA denaturation 2. primer annealing, and 3. primer extensiadamplificatian

These o~erations,which are effected by varying only the temperature, urc repeated n number ol'times to yield amplified products, which are then separated by agurosc gel el~trophoresisand wsualized either by ethidium bromide fluorescence or autoradiography.

Overview Although the theoretical basis of PCR was first enumerated by Kleppe et al. ( I ) , the technique did not arouse general interest until Mullis and co-workers developed PCR into a technique t h a t could be used to generate large

Target D N A + primers+ dNTPs + Mgz. + D N A polymerase

'Correspondence to this author. Current address: Food and Drug Administration, 200 C Street SW, HFS-128, Washington, DC 20204; (202) 205-4920

+..1

-......

-....+

r'

-.....,

1 ,......

1

1

I

Original D N A

PCR primer New DNA

-

+..1

I...

-"1

" " " "

Denature, anneal and synthesize

I

-7

etc.

Denature, anneal and synthesize

I

+..1

="'-1

etc.

Denature, anneal and svnthesize

I

. . . ..

Denature, anneal and synthesize

I

I

I Figure 1. The polymerase chain reaction. The DNA to be amplifiedis first denatured by heating at 95 'C. The primers are annealed to the target DNA in the presence of a DNA polymerase, magnesium ion, and excess dNTPs at a temperature between 37 to 55 T. Amplification/extension is then carried out by raising the temperature to ca. 72 'C. The first cycle generates a product of indeterminate length;the second cycle generates the short product which accumulates exponentially with each successive round of amplification. Volume 70 Number 4 April 1993

273

ference between the primers versus denatured DNAfavors t h e formation of a primer-template complex over reannealing of the two DNA strands. The primers, now annealed to their target sequences, are then extended with DNA polymerase a t 72 'C. The cycle of denaturation, annealing, and DNA synthesis is then repeated (Fig. 1).The products of one round of amplification serve as templates for the next round; thus each successive cycle essentially doubles the amount of desired material, resulting in the exponential accumulation of the target DNA.

Taq DNA Polymerase A DNA polymerase is a n enzyme that synthesizes new DNA strands o ' m a primed template strand. The original protocols for PCR used the Klenow fragment of DNApolymerase I from E. cnli to catalyze the extension of the annealed primers. (Klenow is the large fragment of E. coli DNApolymerase I). Amajor drawback of using the Klenow fragment was that it is inactivated at the temperature necessary to denature DNA. Thus each reaction cycle required the addition of a fresh aliquot of enzyme. The use of a thermostable DNApolymerase (Taqi, originally discovered in a hot spring in Yellowstone National Park and purified from the thermophilic bacterium Thermus aquaticus, eliminated this problem. Taq DNA polymerase can survive extended incubation a t 95 'C and its activity is not substantially reduced during the repeated denaturation steps. Mispriming (incorrectly annealed primers and misextension of incorrect nucleotides (nt))is also greatly reduced since the annealing and extension steps are camed out a t higher temperatures. This results in a substantial improvement in the specificity and yield of the amplified product. In theory, the amplification yield is exponential: 2", where n is the number of cycles. A 30-cycle amplification would be expected to produce lo9copies of new DNA. In practice, however, the overall amplification is limited by the amplification efficiency of each cycle. After a certain number of cycles, highly dependent upon individual reaction conditions, the amplified product stops accumulating exponentially and enters a linear or stationary phase. This is known as the reaction plateau. A variety of experimental parameters, for example, the Tq concentration, which becomes rate limiting, cause reaction plateau. The efficiencyof amplification has been estimated to be between 60 to 85%. This results in a yield of (1 + efficiency)"

which translates, for a 30-cycle amplification, into lo6-los copies of new DNA, which is significant nevertheless. But the true power of PCR can be discerned by examining the initial quantity of template DNA necessary for PCR. Typically, between 0.05-1.0 pg of single copy geuomic targets,

as long as 2-10 kb in length, can be obtained from a 3 0 3 5 cycle amplification from 1pg of starting template DNA. . A limitation of T w i s i t s e r r o r rate. or r a t e of misincorporation of n&leotides (nt), which.results from the enzyme not havinc 3'-5' exonuclease (proofreadine) activity. Recently two new DNA polymerases have become available, both of which possess 3'-5' exonuclease activity. Vent polymerase and v u polymerase, isolated from Thermococcus litoralis and Pyrococcus furiosus, respectively, .. were discovered in geoth&nal vents on the ocean floor. These enzymes have error rates severalfold less than T w polymerase. Regardless, individual error rates vary ac; cording to the DNA sequence to be amplified and variations in reaction conditions. For most applications the error rate will not be a problem. Variations of PCR Amplification of RNA

RNA may be obtained from cells via standard protocols such as guanidinium isothiocyanate/CsCl gradient (3).The enzyme reverse transcriptase (RT) is then used to prepare complementary DNA (cDNA) prior to conventional PCR (Fig. 2). Two types of RT are commercially available: a form isolated from avian myeloblastosis virus (AMV) and a recombinant form from the RT gene of the Moloney Murine Leukemia Virus (MMLV). Both forms lack 3'-5' exonuclease activity This is quite important as i t indicates that RT is prone to misincorporation errors; in the presence of high concentrations of dNTPs and M e , t h e misincnrporation rate is about 1in every 500 bases. Regardless of which method is used to isolate RNA, a finite but nominal concentration of DNA will be present. Therefore, prior to the RT step, it is advisable to treat the RNA with the enzyme DNase I. DNase I is a n endonuclease that hydrolyzes DNA a t sites adjacent to pyrimidine nucleotides. In the presence of M e , DNase I attacks each strand of DNA independently; the cleavage sites are distributed in a statistically random fashion. In order to check on the effectiveness of the DNase treatment, a no-RT control should be included and subsequently amplified to check for the presence of amplified DNA. Anchored PCR

One disadvantage of ordinary (symmetric) PCR is that the sequence at the end of the DNA frapments must be knownin order to construct the appropriate primers. This is often a limitation since the sequence of a s~ecificeene may not be known. In this case a novel techniq;e knoin as anchored I'CR may be of use. Anchored PCR involves addingan artificial primer, such as a poly {dG)tail, to one DKA strand with the enzyme terminal deoxynucleotidyl transferase (TdT)(Fig.3). TdT is a primer-dependent DNA poly-

- -First strand cDNA

'3-'5 4..

. . .. .-

5'

m . .. . .b

Second strand cDNA synthesized PCR components

added

-+

Amplify

Reverse Transcriptase Figure 2. Amplification of RNA. The RNAis copied into a cDNA by the enzyme reverse transcriptase.ordinary (symmetric)PCR is then preformed on the double-strandedcDNA. 274

Journal of Chemical Education

merase t h a t catalszes t h e repetitive addition of deoxynucleotides to the 3' terminus of DNA. The template is then amplified with a specific primer and a second primer consisting of, in this case, p l y (dC), the anchor (4).

symmetric PCR on ds-DNA, isolating and purifying the products, followed by reamplification with a single primer. This asymmetric reamplification would supply a sufficient quantity of ss-template for dideoxy sequencing.

lnverse PCR Inverse PCR is used to amplify DNA outside a known sequence. The method involves digesting the DNAwith restriction enzymes and linking the ends ofeach fragment to form a circle (Fig. 41. The circle then contains the known sequence flankingit. Pruners are then bound to the known sequence. However, they are designed so that DNAsyntheS that is. DNA svnthesiti takes the sis D ~ O C C C ~outward: long away around the circle to the ocher primer, rather than inward, a s in conventional PCR. The first round of amplification yields a linear product in which the sequences are inverted: the unknown sequences are bracketed by primer sites and ordinary (symmetric) PCR can begin (5).

Region containing known sequences

s-DNA

Restriction enzyme site

V

A

4

Digest

Ligate

Asymmetric PCR Asvmmetric PCR is used to amnlifv a snecitic strand of DNAby using a n unequal, or asymmetric, concentration of primers. During the initial thermal cycles, exponentially derived (double-stranded) ds-DNAaccumulates. As the low concentration primer becomes depleted, further cycles generate a n excess of one of the two strands, depending on the limiting primer. This (siugle-stranded) ss-DNA accumulates linearly and is complementary to the strand previouslv amplified bv the limiting primer. ~ s y m m e k i cPCR is less efficient than standard PCR; hence, more cycles (30-40) are required to obtain a maximum yield of ss-DNA. Primer ratios lie in the 50-100:l range. The concentration of target DNA is important; yields will be low if too little is included, while background can be excessive if too much is included. Low ss-DNAvields can be increased by carefully increasing the ?Zlq concktration: lower dNTP concentrations, which minimize mispriming at non-target sites, are used to limit backA

"

.

--

orn.,na *. .. .

The major problem with asymmetric PCR has been that the amount and quality of template differs frnm preparation to preparation. his depends primarily on the-primers and target DNA. Many empirical studies may be necessary in order to produce good ss-templates. In addition, asymmetric PCR does not always result in reproducibly high yields of ss-product. This can be overcome by performing

DNA

3'

S

s

3.

TdT

GGGG

dGTP

Figure 4. Inverse PCR. Double stranded-DNA is digested with a restriction enzyme and the ends are linked together to form a circle. Primers, designed so that DNA synthesis proceeds outward from each primer, are then annealed to the known sequences. A second restriction enzyme cleavage within the known sequences results in a linear template with primers located at the termini. Ordinary (symmetric) PCR is then performed. Addition of anchored polyC as second primer

Addition of poiyG tail I C C C C GGGG

4

...

, -+ Amplify

*....I

TdT: terminal deoxynucleatidyl transfsrase dGTP: deoxy Quanosine triphosphate F~gure3 Anchoreo PCR A polyG la' ~senzymatcal y aaaed oy TdT to one strand ol DhAconla8nmg unknown sequences Tne anchor,consfstlng of one pruner oonded lo polyC, s then adoea. PCR 1s then performed in tne normal manner Volume 70 Number 4 April 1993

275

Multiplex PCR Multiplex PCR refers to the simultaneous amplification of different eenomic regions durine .. ., a single PCR. Chamherhin et al. pioneered multiplex PCR for the diagnosis of Ihchenne Muscular 1)vstroohv (DhlDi 16,. DMD IS a neuromuscular disorder &at reshts from mutations at the human dystrophin gene; it is among the most common human genetic disorders, affecting approximately 1 in 3500 male births. One-third of all cases arise via a new mutation; of this amount, about 60% are associated with DNA deletions. Deletions at the dystrophin Eerie are commonly detected by restriction enzyme digestion followed by Southern blotting. This method results in more than 65 DNA fragments requiring hybridization (hydrogen hondingof probe sequences to target sequences) with 7-9 cDNA nmbes. which is clearlv unsuitable for routine use in clini~~~~~-~ ~ - ezlabbratories. ~ u l t i & e xPCR is capable of detecting 8090% of all dystrophin gene deletions. The technique relies on the observation that dystrophin gene.deletions are concentrated in two specific regions of the 2 megabase DMD gene. Consequently, only a small percentage of the gene needs to be analyzed. Nine primer sets are used in the reaction. The PCR products are analyzed by gel electrophoresis. Deletions correlated with the disease are easily identified by the absence of expected DNAfragments. Analysis of 150 patients bv multiplex PCR was confirmed by Southern andysis; onfy two Eases required further analysis. A recent multicenter clinical study determined that multiplex PCR detected 82% of the deletions detected by Southe m blotting, with a false positive rate of 0.013% (7).

-

Quantitation In order to understand gene expression it is necessary to accuratelv measure messenger RNA (mRNA) concentrations. ~ifferencesin mRNA expression can provide information for the diagnosis of infectious disease or cancer. Conventional methods (Northern analysis) are not sensitive enough to detect low copy number mRNA. Hence PCR should be ideal for quantitation. However, quantitation is difficult due to the exponential increase of PCR product per amplification cycle:~mslldificrences in any one experimcntal variable meatly affcct PCR product yield. Several experimental approaches can be employed to overcome these limitations. The most obvious approach is to use an internal control in the same tube prio; to amplification. This results in the normalization of amplification variables such as M e and dNTP concentrations, cycle number, temperature, and length of cycle steps. The internal control also verifies whether a result is a true falsenegative if no signal is obtained from the sequences of interest. Of course, the internal control PCR product should be different enough in size to be resolved from the product of interest. Wang et al. constructed a synthetic gene to use as an internal control (8). They cloned (inserted) into a control plasmid (circular, extra-chromosomal bacterial DNA) the 5' primers of 12 target mRNAs, followed by a polylinker (an artificially constructed sequence of restriction enzyme sites), then followed by the 12 complementary 3' primers. Placement of the polylinker between the 5' upstream and 3' downstream phmers allowed for the cloning of primer p a r s for other genes. Prior u, PCH, the plasmid was linearized a t a restriftion enzvme site and then transcribed with %polymerase. This resulted in a cRNA, which was reverse-transcribed and am~lifiedwith the target mRNA in the same tube. Thus the'plasmid ultimateli served two functions: ~

~

1. it served as the internal mRNA wntrol for the reverse transcription reaction, and

276

Journal of Chemical Education

2. it was used to generate a standard curve for quantitating the target mRNAs.

Differences in orimer efficiencywere minimized since the samc primer sit was used for both tcinplates. In addition, tuhe-to-tube variations were eliminated since the quantitation step was performed in the same tube. The Gntrol plasmid,. ~AW109. is commerciallv available. . The above method was applied to quantitate several lymphokines (soluble protein factors secreted by lymphocytes) after stimulating human macrophages with bacterial endotoxin. PCR was performed on total cellular RNA with radiolabeled primers. After electrophoresis of the products, the amount of radioactivity from the excised bands was plotted against either template concentration or cycle number. Relative to the internal control, they determined that 1 ng of RNA contained lo4 molecules of interleukin la ( I d l a ) . When plotted against cycle number, the rate of amplification was found to be exponential between 14 and 22 cycles; further amplification resulted in a decreased rate. The efficiency of amplification, calculated from the slope, was found to he 88% for both the template and the standard. Several other mRNA species were analyzed before and after stimulation. The concentration of IL-la and IL-1D mRNA increased 50-fold after stimulation. The levels of platelet derived growth factor A (PDGF-A), macrophagecolony stimulating factor (M-CSF)and tumor necrosis factor increased 5-10-fold, while PDGF-B mRNA remained constant. Quantitative PCR performed by this method thus provided a detailed picture of the transcriptional activity in both resting and induced states in human macrophages. One of the problems with the above method is that it deoends both ~ - on ~ ~ the unknown temolate and reoorter cDNA as having the same efficiency of &plification:~n this case, however, the two amplification reactions differ in the dissociation temperature of t h e DNA templates and primerltemplates along with the length and concentration of the DNA template. As such, amplification efficiencies of the two unrelated cDNAs may be different due to different initial reaction rates, differential decrease in reaction rates, and different amplification efficiencies . per cycle. . Gilliland et al. developed a n alternate technique to overcome these limitations (9).It involved co-amplification of a competitive DNA template and a target DNA template with identical primers. The competitive template was identical to the cDNA of interest except for a mutated restriction site. Thus the ratio of product to competitor remained ~ - --- ~ constant throuehout the amolification. reeardless of any changes in cyclenumber or &action vaAabres. The rate should thus reflect the initial concentration of the unknown cDNAversus that of the competitor cDNA. One limitation of this method is that the efficiencyof reverse transcription is assumed to be 100%;if it is not, the amount of actual mRNA present will be underestimated. As an example, granulocyte-macrophage colony stimulatingfactor (GM-CSF)mRNAwas quantified from a stimulated cell line using genomic GM-CSF (gDNA)as the competitive template. Both the cDNA and gDNA should be amplified with the same efficienciessince the primers are identical. In effect, the gDNA and cDNA templates comnete for PCR substrate. Quantitation was achieved bv ti;rating an unknown amount of cDNA against serial klutions eDNA concentrations. Northern analvsis ~ - ~ of - known - ~ could not detectYanyGM-CSF mRNA from less than.lo6 cells. Competitive PCR detected GM-CSF mRNA, in wncentrations ranging from 0.47-0.55 amol, in as few as 200 cells. This value corresponds to each cell having about 1500 GM-CSF molecules.

.~

Applications of PCR Sequencing PCR Products

Didww chain termination (Sanger) is one of the most commo& used methods for DNA sequencing. Either sinele-stranded or double-stranded temolates may be used: Lowever, the best results are obtained from single: stranded templates, which require the use of an MI3 sequencing vector. In order to use M13, which is a singlestranded DNAE. wli bacteriophage (a bacterial virus), the portion of DNA to be sequenced must be cloned into the vector. Asequencing primer is then annealed to the vector and the sequencing protocol initiated. While PCR can be used to amplify specificfragments, each fragment must be cloned into M13, which is time-consuming. A better technique would be to sequence PCR products directly. In addition to simplicity, direct sequencing would greatly reduce the potential for errors due to imperfect PCR fidelity. An ideal method to eenerate simle-stranded temdate would be with asynun&ic PCR. the single-stranded template could then be sequenced with either the limiting primer or a third internal primer. Of course, two separate asymmetric PCR reaction would be necessary in order to sequence both DNA strands. Gyllenstein and Erlich used asymmetric PCR to sequence a portion of the major histocompatibility complex (MHC) (10). The MHC consist of a family of surface cell glywproteins; they are also known as HLA (human leumcvte antieens) as thev were first discovered on leucocvtes (khite bl;od cells). Ti;ese highly polymorphic proteinsare involved in specific immune responses. Knowledge of the specific alleles (alternate forms of a gene) within this region are useful in tissue typing for transplantation to minimize graft rejection by selecting HLA-matched donors and recioients. HLA twine "- is also useful in forensic science andbaternity determinations. In order to study polymorphisms, a particular gene must be examined in a population of individuals. In this case asymmetric PCR was employed to examine the sequence diversity of the HLA genes. Following amplification and oroduct clean-uo. the am~lifiedDNA was seauenced with o f thk 25 individuals ex&ned, eight the limiting seauences were identified as allelic variations within the HIA genetic locus. This sequence information can then be used to desim allele-s~ecificoligonucleotideIASO)probes; that is, proges that recognize single base-pair mutations. Once the allelic sequences are known, their frequencies in patient and control populations can be determined in PCR amplified DNA by dot blot hybridization with the AS0 probes. (Dot blot hybridization involves the transfer of spots or dots of amplified DNAto a nitrocellulosefilter; the spotted filter is then hybridized with a labeled probe.) Dideoxv seauencine from double-stranded temolates ) become increasingly populk The (i.e., plasmid ~ N A h& advantaeesof this method are that either DNAstrand may be sequenced and the MI3 cloning step is eliminated. HOGever, prior to sequencing, the double-stranded template must be denatured. The trick is to maintain sufficient dissociation of the rapidly - - reannealing strands during the annealing and extension of the sequencing rimer. Strand reassociation can be slowed by quickly cooling the denatured template on ice. Double-stranded sequencing is particularly useful for screening for specific plasmid constructs &om ~ N ~ m i n i ~ r e ~ a r & , i -o n s . Recent efforts have been directed toward the development of direct PCR double-stranded sequencing protocols. Several oroblems. none of which are insurmountable. persist with double-stranded sequencing. ARer the PCR is oerformed, the amolification primers. buffer, and dNTPs must be removed from the PCR prod;cts before sequenc-

ine. In oarticular. this eliminates anv comoetition between th; amplification and the sequencing priker(s). Removal can be effected either by anion exchange wlumns or excision of the appropriate bands from an agarose gel. Asewnd oroblem is that small. linear. PCR-generated framents tend to have regions df secondary s'ucture that L P e d e the wlvrnerase as it *oroeresses alone - the temolate. This " can be overcome either by rapid cooling after denaturation oolvmerase. In this latter case. or bv seauencine with Taa.. rea&ionAtempe~atures between 55-75 'C will eliminate most of the sewndanr structure of the templates. A recent advance in this area involves multiple cycles of linear PCR (LPCR) (11).After svmmetric PCR is oerformed, the products are isolated-and subjected to sequencingprotocol that uses Taq polymerase. The reactants are cycled through a 95 'C denaturation step, a 55 'C annealing step, and a 72 'C extension step for about 20 cycles. This results in a linear accumulation of sequenced templates; in effect, a population of sequenced templates is generated. As a result, femtomole (1fmol = 10-15 moll quantities of template can be used to generate up to 500 bases of readable sequence. An additional advantage is that the 95 'C denaturation step limits both template reannealing and secondary structure. Tag DNA polymerase cycle sequencing kits are available from several vendors.

.

-

a

Application to AIDS

Human immunodeficiency virus (HMis the etiological agent of acquired immune deficiency syndrome (AIDS). HIV is a retrovirus: genetic information is contained in RNA rather than DNA. The H N genome cames the enzyme reverse transcriptase (RT) which catalyzes the synthesis of viral RNA into cDNA. This process is camed out in the cytoplasm of the infected cell. The cDNA, or proviral DNA, may exist either as an unintegrated form in the cytoplasm, or it may be translocated to the nucleus where it is integrated into the genome of the infected cell. HIV infection can be confiied by several immunological assays, including tests that detect antibodies made against the viral proteins, that is, the Westem blot (WB) assay. However, Gery few peripheral blood cells contain proviral DNAearly in HNinfection and there is a variable period of time when an individual is infected and when anti-HN antibodies can be detected (usually about 6 months). The potential of PCR to identify proviral DNA was quickly realized and has since been widely employed in basic research and clinical studies. The most imoortant criterion for H N analysis by PCR is identification of regions of the viral genomgfor amplification. H N has a high demee of genetic variation; only invariant reg.lons of the genome that lack significant secondary structure can he usefully targeted for amplification. ~ i ~conserved h l ~ sequences ha;e been identified and primers and probes for these regions have been designed (12).With this information. PCR has been used to coniirm the presence of proviral DNA in seropositive tanti-HN antibodies ~-~~~~~~in sera1 individuals in all staees of disease (1.7). It has also detected proviral DNA in hGh-risk seronegative individuals orior to seroconversion (14) and has detected unintegratei viral DNA in brain tissue, which has been hvoothesized to correlate with the oathogenesis of H N encephalitis (15).Conversely, PCR has detGcted HIVviral sequences in seropositive asymptomatic individuals who were negative by other detection assays (16). A n important application of PCR has been in the identification of HN-infected infants. Infants born to HIV-infected mothers have maternal antibodies acquired transplacentally and thus are seropositive. Maternal antibodies &ay circulate up to 15 months in newborns making it dif~~

~~

Volume 70 Number 4 April 1993

277

Two experimental techniaues are emdoved . - in DNA fingerprinting: restriction fragment len#h polymorphism (RFLPJand PCH-based twinrr methods. RFI.Ps are different DNAfragment l e n d s generated by the action of a restriction enzyme. The fragments are electrophoretically separated, transfemed to a membrane (Southern blotting), hvbridized to a radioactive cDNA probe, and visualized via a;toradiography. The original set'of fingerprinting probes was developed by Jefferies et al. (241.The human aenome contains a number of tandem repetitive minisatehe regions that have extensive allelic variation. These regions also share a common core repeat sequence. Hybridization of DNA to a cDNA probe, consisting of multiple copies of the core sequence, produces a DNA fingerprint unique for each individual. PCR-based methods involve the use of PCR amplified DNA with allele specific oligonucleotide (AS01 probes. AS0 probes will only hybridize to sequences that match it perfectly; a single bp mismatch is sufficient to prevent hybridization. Of course, the DNA sequence of all allelic variants must be known to design the appropriate AS0 probe. Once this has been established, the presence of specific alleles can be examined by dot blot hybridization. Saiki et al. reversed the dot blot hybridization procedure; that is, they attached AS0 probes to the nitrocellulose support and hybridized the amplified DNA to the individual probes (25). Schnittman et al. developed an amplification scheme caUsing this procedure an entire series of sequences could be pable of detecting H N specific mRNA by RNAPCR (21). In analyzed by a single hybridization reaction. An additional this way it was possible to study the in vivo state of H N advantage was that the AS0 probes were sficiently senexpression in HN-infected individuals. After RT of HNsitive to use nonradioactive methods of detection. specific mRNA's. the cDNA was amplified with primers of individual genes. ~ ~ ~ r o i m a t e l y The development of PCR-based DNA typing represents sbecific for the &A'S 84% of the patients had onaoinrr HIV transcription as evione of the more recent advances in forensic science. PCRdenced by the detection of a t leak one ~ N - s ~ e k fmRNA. ic based methods have several advantages over RFLP methThis frequency was observed whether the patients were ods. First, they are easier to perform, do not necessarily asymptomatic or had already developed full-blown AIDS. require the use of radioactivity, and are amenable to automation, which can reduce operator error and increase samPCR has also been used to quantitate proviral DNA to ple throughput. A second advantage is sensitivity; DNA determine the number of infected cells in HIV-positive incan be extracted from single human hairs as well as small dividuals. Quantitation was achieved by serial dilution amounts of blood, semen, and saliva stains. In addition, aeainst a standard amount of infected cells prior to a m ~ l i sample analysis can be easily repeated since PCR-based fication. In seropositive individuals approximately 1 in lo4 methods consume so little sample. T-cells which exoresn the CD4t surfuce molecule (the HTV receptor) contaked proviral DNA. In AIDS patients the At present only a few DNA marker systems based on ratio was 1in 10' CD4+ cells (22). These results are imPCR are sufficiently well characterized for use in forensic portant since infection with H N results in the selective applications. The first and most well-developed system is depletion of the CD4+ T-cell subpopulation. Aoki et al. the HLA-DQa (a subset of the HLA region) system. The studied the distribution of proviral DNA in peripheral specific sequences of this region, previously determined by blood mononuclear cells (PBMC)after fractionation into B Gyllenstein and Erlich (101, were used to design PCR and TceIls(2.?1.PCR analysis showed that 944 o f H N proprimers and AS0 probes. A 242-bp segment of the HLAviral DNA resided in the T-cell fraction and 4% res~dcdin DQJ region was amplified. Reverse dot blot hybridization the B-cell fraction. There was a significant decrease in HIV with AS0 probes identified six alleles defining 21 genoproviral DNA after treatment with the recently FDA aptypes with population frequencies ranging from 0.005 to proved antiretroviral drug 2',3'-dideoxyinosine (ddI). For 0.15. The discriminating power (DP) of this typing system, example, one patient's proviral DNA decreased about 75% that is, the probability of distinguishing between two indiover a 15-week treatment. viduals chosen at random, is 0.93. This compares favorably with the discriminating power of traditional markers such as ABO blood groups with a D P of 0.60. The HLA-DQa-typDNA Fingerprinting ing system is currently used for forensic applications and is commercially available as a kit (26). DNA fingerprinting refers to the characterization of a portion of an individual's genome. Everv individual has a characteristic phenotype-because eve& individual posThe Human Genome sesses a unique hereditary composition (except identical The Human Genome project is an international research twins who have identical DNA sequences). To put this into effort to catalogue and analyze the estimated 100,000 perspective, the human genome contains 3 x 10' nt, which encode information for about 100,000 genes. Given this human genes that lie within human DNA. The ultimate amount of information, it is not surprising that considergoal is to determine the complete sequence of the entire able genetic variation is found from person-to-person. This human genome. Continuously evolving technologies, not uniqueness is defmed by genetic markers inherited from necessarily in existence today, will be used to obtain this information. Knowledge of the human genome and how it one's parents. These genetic markers can be analyzed a t functions will broadly impact all aspects of biological and the level of either protein variation or DNA sequence varimedical research. ation.

ficult to differentiate between maternal and infant antibodies. Recent PCR-based studies have shown that approximately 2040%children born from seropositive mothers are infected (17). About 6000 HN-infected women give birth in the United States each year, and thus a minimum of 1200 children become infected in a year's time. Rogers et al. detected proviral DNA in five of seven neonates (1-16 days old) who later developed AIDS (18)while H N DNA was detected in 20 of 50 infants born to HN-positive mothers (19).These results are important since AIDS develops more rapidly in infants and children and early identification allows for corrective therapies. Serological assays are used to screen blood and blood products for the presence of anti-HIV antibodies. Samples that are repeatedly reactive by ELISA determination are retested with the WB assay; although sensitive, WB assays can produce indeterminant results. Individuals with indeterminant WBs are either HIV-infected and in the early stage of seroconversion or truly HN-negative. A s to be expected, indeterminant WBs result in the rejection of blood from blood donors. The use of PCR for direct detection of H N is therefore more desirable. In one study, 20out ofa total of 100 blood donors had indeterminant WDs. PCR analysis demonstrated that all 20 were HIV negative (20).

-

278

Journal of Chemical Education

The human genome project will probably be performed in two steps. First, detailed maps will be constructed covering the entire genome. Three types of maps will be constructed: cytogenetic, genetic, and physical. Cytogenetic of maps are based on the distinctive bandine- pattern . stained chromosomes when examined microscopically. They are low resolution maps delineating specific genes or specific chromosomal refions. Genetic, or linkage maps, reflect the relative location of genetic markers, such as a physical trait or a particular medical syndrome, by their patterns of inheritance. Physical maps, measured in base oairs. ~,consist of ordered landmarks at known distances. For example, a low resolution physical map would be chromosomal bandine oatterns: a hieh resolution map would be the complete ;;cleotidesequ&ce of the chrombsomes. All of this information will then be assimilated and employed to sequence the individual segments corresponding to all known map *~ositions. PCR techniques will more than likely be involved in even aspect of the proiect. Much of the current discussion invoking PCR inv6lv& the use of sequence tagged sites (STS) as landmarks for the construction of genetic and physical maps (27). The basic premise of this concept is that all DNA fragments used to construct these maps would be identified by a 200600 bp site unique to that fragment. Each site would be sequenced, and primers would be designed to enable specific PCR amplification a t that site. Thus PCR could be used to regenerate a DNA fraement from anv eenomic reeion. The final product from ~ ~ ; S T Sproposai~ouldbe a >TS map of tbk human genome. The map would be a composite of both genetic and physical maps, with genetic markers and physical landmarks translated into STSs and ~ositionedrelative to each other. Individual laboratories could use whatever mapping techniques they chose; results would always be reporiid 6 terms of the STS markers. Adatabase would be generated containing the location, sequence, and primers necessary to amplify each STS. The database thus would be laboratory independent. The STS approach would eliminate the need to physically store clones andlor distribute them to laboratories worldwide. Finally, it would provide a common language and landmarks for mapping (28). The high resolution physical map may require as many as 10,000 polymorphic DNA markers. One approach for the generation of these markers involves primers directed to interspersed repeated sequences (IRS PCR). IRS PCR involves using primers directed to Alu sequences (Alu PCR) to amplify human DNA falling between Alu repeats in the genome. The Alu sequence is a tetranucleotide that can be cleaved by the restriction enzymeAlu I. This family is the most common short interspersed repeat sequence (SINE) in the human genome, with one 300 bp sequence occurring approximately every 6 1 0 kb. Alu PCR was originallv devised for identifvine human DNA in human-rodentehybrid cell lines. ~ o i e i e rit, has demonstrated to be from soecific chman efficient tool for eeneratine- orobes . mosomal regions (297. The IRS concept can be extended to other sequences. This is actually quite important due to the uneven distribution of other repeated sequences throughout the human genome. For example, Kpn, a long, interspersed repeat sequence (LINE:, recognized by the restriction enzyme K p n I, is the second most common repeated sequence i n the human genome. The Alu a n d K p n sequences represent more than 90%of all middle repetitive elements within the human genome (30). At present, optimizing IRS-PCR to develop probes from any targeted chromosomal region is limited by knowledge of repeat sequence distribution within the human genome. Thus a variety of primers and

.~~

~~~~~

~

~

&

methods involving PCR will need to be developed to obtain uniform coverage of the entire genome with DNAmarkers derived bv IRS-PCR. Other applications of PCR to mapping and sequencing include chromosomal walking and expansion of yeast artificial chromosome WAC, clones (31),mapping and analysis of expressed sequences from particular genomic regions (321, and direct PCR-coupled DNA sequencing (33). Review Articles There are several recent review articles worth noting. Erlich et al. describe recent advances in PCR (34). while Bej et al. (351, Lizardi et al. (361, and Gibbs (37) provide general overviews; a summary of nucleic acid amplification strategies has also been presented (38).Bloch has presented an in-depth discussion of PCR reaction principles (39). Wetmur has summarized the use of cDNA probes for hvbridizations (40). . . PCR seauencine methods have also been reviewed (411;optimum conditions for ds-sequencing have been reviewed 142.43). Methods for the auantitative determination of m ~ ~ ~ c o n c e n t r a t ihave o n s been summarized (44). Kumar presented a detailed experimental protocol for PCR (45). Muul(l2) and Poiesz et al. (46) discuss PCR for the detection of AIDS; both include experimental protocols. Reynolds and Sensabaugh discuss PCR-based forensic analysis (47). Finally, Linz discuss several experimental strategies for optimizing PCR (48). Summary

PCR has revolutionized recombinant DNAtechniques by permitting the generation of almost any nucleic acid seouence in vitro. Mani~ulationof these amnlified seauences gas broadly impacte'd basic and clinicai sciences: Other amplification strategies, aside from those discussed, are possible. Future refinements in PCR technology will involve easier auantitation. automated svstems for better reproducibility, and overall improvements in methodology in order to achieve routine analyses. Acknowledgement WCT acknowledges Peter Bressler, National Institutes of Health, for introducing him to PCR techniques. Literature Cited 1. meppe, K.;OhtauLq E.; Kleppe, R.;Molinelu, ; lOvol~vo;H. G. J Mole. Bial 1811,

5. 6.

Tnglia,T,:Pdersan,M. G.;andKemp,D.J.NudeleAeidsReg. IW.16,8186. Chamberlin.J.8.;Cihhi, R A,; h i e s J. E.; Nguyen, P. N.; CasLey, C. T. Nvclplc driZ.Peo

7. 8. 9. 10. 11.

1IEllldl ----, .-,-..

Chamberlain, J. 9 , et al. JAMA 1882,267l191,2609. Wang, A.M.; Doyle, M . V; Mark, 0. F R o s Not Amd Sci 1963.86,9117. Gilliiand, G.; Perrin, S.; Blanchad, K . P m NoLAmd. Sci. 1930,87,2725. Gyllenstein, U.;Edich, H.Pmc. Not. A d Sci. 188485,1652. Murrsy. V . Nudaie Acids. Res. 1989.17.8869.

RT.;schochetm&,C.;Epstein, J.E.J Clin. I m m u m w I W , 1 1 , 1 6 1 . 15. Pang, 8.; l(oyanagl,Y.;Miles, S.;Wilily, C.: Vinters, H.V.;Chen,I. S. YNolvre ISSO, 343.85. W.;Maek,D.H.; Sninsky.J.J.;Krebs.J.W.; Feorina, 16. Ou,C. Y.; Kwak, S.;MiW1ell,S. P; Warfleld,D.;Sehmhetman,G. Sc&ncolW,238,296. 17. Lsure, F;Courgnaud,V.;Rousiou., C.;Blanche,S.;Vebeq E; B u g a d , M.;J a r n e t , C.;Dnacelli, C.; Breeholt, C. h n e t 1988,11,538. J.Md. 1888,320, 16. Ragers,M.F;ou,C.Y.;Rsyfield,M.;momaa,P11:etal.N.Eng 1649. 19. Laehe, M.; Maeh.B.h n a t l988,11,418. . K.; Jet", B. W.; Hewlet", I. K.;Epstein, J. S; Al&, H.J. 20. Genesea, J.; Shih, J. W h n n t 1989,11,1023. F:Fsuci,AS.AIDSRpa. 21. Schnithnan, S. M.;Greenhouae. J.: h s , H .C.;R-.P & H u m R~fmuirusos1991,7,361. 22. Spector, S. A.; Hsia, IC, Denam,E;Spetn, D . H. Clin. Cham. 1888,35,1~%1. 2s. &~,s.;Yarchaan,R.;momaa,RV.;Pluda.J.;Marayk,K;Bmder,S.;Mitauya,H. AIDS fis. &Hum.Refmvirwos 1990,6,1331.

Volume 70 Number 4 April 1993

279

24. Jeffffriris, A J.: Wilson, V;Thein, S. L. Nature 1985,314(60063,61. 25. Ssiki, R. K: Wslah, P S.; Levenson,C. H.;Erlieh, H. A. P m e Nor A d Sei. 1888, 86,6230. 26. Erlich, H. A,, E d P C R Toehnology, SStoeMon Prese: NenYork, 1990. 27. Olsen, M.; Hwd, L.: Cantor, C.; Botstein, D. Sciena 1888,245,1434. 28. Rose, E. A. FASEB J. 1991,5,46. M .F.;Ramii.SoIk, R.; Wwbster, 29. Nelson, D. L.; Ledbetter, S. A: Cccb.Lbo;V~ctoria, T. D.; I d b e t t e r , D. H.; Caskex C. T Pme. Not, puu* Sci. 1989,86,6686. 30. Maysiz, R. K,Tomex D. C.; Meyne, J . M.; Buckngham, J. M.; Wu,J. R.; Burka, C.: S h t k i i , K. M.; &ad, W B. Denom& 1989.4.273. 31. Green, E.D.; Olsen, M. Y P m c Not.Amd.Sei. 1990,67,1213. 32. Corb, L.; Maley J. A.;Nelson, D. k Caakey, C. T. S e h m 1990,249,652. 33. Inn&, M.: Myambo. K B.; Gelfand, D. H.; Brow, M. A. Pmc. Not. A c d Sci. 1988, 85,7652. 34. Erlich, H.A,; Gelfand, D.; Sninsky, J . J . Science 1991,262,1643. 35. ~ e jA., K.; ~ a h b v b a n M. i H.; ~ t l a sR. , M cpitiml R ~ U . ~ i ~ hM a O~~ ~i. o l 1981, . 26,301.

280

Journal of Chemical Education

36. Lkerdi, P M. aod Krsmer, F.R., %.n&Bid. Sci. l391,9,53. 31. Gibb8.R A.,Anal. Cham 1990,62,1202. 38. Anonymous. J oJNIHRea. 1991,3,81. 39. Bloeh, WBioeham 1981,30111),2135. 40. Wehnur, J . G. CrillmlRev Bioeham Molc BioL 1981.26211. 41. Gyuenatein. U.Bioechniqrws 1363,7,IW. 42. Casanova, J.; Wnnetier, C.; Jsnlin, C.; Komilsky, P N&k AeGA Res. 1880, IS, 4028. K.: Kato.1.; Shuao,T.Biofschniqu~s1990,9(1)66. 43. Kua~a~a,N.;Uemori.T.;Asadd, 44. Beeker-Andre. M. Molh. Mdc CdlBiol. 1991.2, 189. 45' Kumar'R'Toehnigua 1988'1' 46. Poiesz,B. J.; Er1ieh.G. D.;Ryme. B. C.: Wells, K,Knak.S.;Sninaky, J. Med. Kml. 1980,9,41. 47. Reynolds, R.; S-ab=ugh. G.And. Cham 1981.63.2. 48. Linz,U. Mdh. Mole CellBioL 1981,2,98.